Oligonucleotides have been used for diverse biological purposes including antisense inhibition of gene expression, PCR (polymerase chain reaction), diagnostic analysis by gene chips, and so on. Since oligonucleotides interact in a sequence specific manner with nucleic acids such as DNA and RNA, they are quite useful to predictably modulate biological processes involving DNA or RNA within cell. Unlike small molecule drugs, however, oligonucleotides do not readily penetrate mammalian cell membrane, and therefore hardly affect biological processes within cell unless properly modified or formulated to readily penetrate plasma membrane.
Proteins as Drug Targets:
Proteins mediate diverse cellular functions. It would not be surprising to find that most of currently marketed drugs show therapeutic activity through modulating functions of protein(s). For example, non-steroidal anti-inflammatory drug aspirin inhibits enzymes called cyclooxygenases for its anti-inflammatory activity. Losartan binds to and antagonize the function of a trans-membrane receptor called angiotensin II receptor for its antihypertensive activity. Rosiglitazone selectively activates an intracellular receptor called peroxisome proliferator-activated receptor γ (PPARγ) to elicit its antidiabetic activity. Etanercept is a fusion protein which binds to a cytokine called tumor necrosis factor-α (TNF-α), and neutralizes the biological activity of TNF-α for its anti-rheumatic activity. Herceptin is a monoclonal antibody to treat breast cancer by selectively binding to erbB2 over-expressed in certain types of breast cancer cells.
Antisense Inhibition of Protein Synthesis:
Proteins are encoded by DNA (2-deoxyribose nucleic acid). In response to cellular stimulation, DNA is transcribed to produce pre-mRNA (pre-messenger ribonucleic acid) in the nucleus. The intron portion(s) of pre-mRNA is enzymatically spliced out yielding mRNA (messenger ribonucleic acid), which is then translocated to the cytosolic compartment. In the cytosol, a complex of translational machinery called ribosome binds to mRNA and carries out the protein synthesis as it scans the genetic information encoded along the mRNA. (Biochemistry vol 41, 4503-4510, 2002; Cancer Res. vol 48, 2659-2668, 1988)
An oligonucleotide binding to mRNA or pre-mRNA in a sequence specific manner is called antisense oligonucleotide (AO). AO may tightly bind to an mRNA and inhibit the protein synthesis by ribosome along the mRNA in the cytosol. AO needs to be present within cell in order to inhibit the synthesis of its target protein. AO may tightly bind to a pre-mRNA in the nucleus and affect the splicing of the pre-mRNA, producing an mRNA of altered sequence and consequently an altered protein.
Unnatural Oligonucleotides:
Oligonucleotides of DNA or RNA are susceptible to degradation by endogenous nucleases, limiting their therapeutic utility. To date, many types of unnatural oligonucleotides have been developed and studied intensively. (Clin. Exp. Pharmacol. Physiol. vol 33, 533-540, 2006) Some of them show extended metabolic stability compared to DNA and RNA. Provided above are chemical structures for some of representative unnatural oligonucleotides. Such oligonucleotides predictably bind to a complementary nucleic acid as DNA or RNA does.
Phosphorothioate oligonucleotide (PTO) is a DNA analog with one of the backbone phosphate oxygen atoms replaced with sulfur atom per monomer. Such a small structural change made PTO comparatively resistant to degradation by nucleases. (Ann. Rev. Biochem. vol 54, 367-402, 1985)
Reflecting the structural similarity of PTO and DNA, they both poorly penetrate cell membrane in most mammalian cell types. For some types of cells abundantly expressing transporter(s) for DNA, however, DNA and PTO show good cell penetration. Systemically administered PTOs are known to readily distribute to the liver and kidney. (Nucleic Acids Res. vol 25, 3290-3296, 1997)
In order to facilitate PTO's cell penetration in vitro, lipofection has been popularly practiced. However, lipofection physically alters cell membrane, causes cytotoxicity, and therefore would not be ideal for long term therapeutic use.
Over the past 20 years, antisense PTOs and variants of PTOs have been clinically evaluated to treat cancers, immunological disorders, metabolic diseases, and so on. (Biochemistry vol 41, 4503-4510, 2002; Clin. Exp. Pharmacol. Physiol. vol 33, 533-540, 2006) Many of such antisense drug candidates have not been successful partly due to PTO's poor cell penetration. In order to overcome the poor cell penetration, PTO needs to be administered at high dose for therapeutic activity. However, PTOs are known to be associated with dose dependent toxicities such as increased coagulation time, complement activation, tubular nephropathy, Kupffer cell activation, and immune stimulation including splenomegaly, lymphoid hyperplasia, mononuclear cell infiltration. (Clin. Exp. Pharmacol. Physiol. vol 33, 533-540, 2006)
Many antisense PTOs have been found to show due clinical activity for diseases with a significant contribution from the liver or kidney. ISIS-301012 (mipomersen) is a PTO analog which inhibits the synthesis of apoB-100, a protein involved in LDL cholesterol transport. Mipomersen manifested due clinical activity in a certain population of atherosclerosis patients most likely due to its preferential distribution to the liver. (www.medscape.com/viewarticle/556073: Accessed on Feb. 19, 2009) ISIS-113715 is an antisense PTO analog inhibiting the synthesis protein tyrosine phosphatase 1B (PTP1B), and was found to show therapeutic activity in type II diabetes patients. (Curr. Opin. Mol. Ther. vol 6, 331-336, 2004)
In phosphoroamidite morpholino oligonucleotide (PMO), the backbone phosphate and 2-deoxyribose of DNA are replaced with phosphoamidite and morpholine, respectively. (Appl. Microbiol. Biotechnol. vol 71, 575-586, 2006) While the DNA backbone is negatively charged, the PMO backbone is not charged. Thus the binding between PMO and mRNA is free of electrostatic repulsion between the backbones, and tends to be stronger than that between DNA and mRNA. Since PMO is structurally very different from DNA, PMO wouldn't be recognized by the hepatic transporter(s) recognizing DNA. However, PMO doesn't readily penetrate cell membrane.
Peptide nucleic acid (PNA) is a polypeptide with N-(2-aminoethyl)glycine as the unit backbone, and was discovered by Nielsen and colleagues. (Science vol 254, 1497-1500, 1991) Like DNA and RNA, PNA also selectively binds to complementary nucleic acid [Nature (London) vol 365, 566-568, 1992] Like PMO, the backbone of PNA is not charged. Thus the binding between PNA and RNA tends to be stronger than that between DNA and RNA. Since PNA is structurally markedly different from DNA, PNA wouldn't be recognized by the hepatic transporter(s) recognizing DNA, and would show a tissue distribution profile very different from that of DNA or PTO. However, PNA also poorly penetrates mammalian cell membrane. (Adv. Drug Delivery Rev. vol 55, 267-280, 2003)
In locked nucleic acid (LNA), the backbone ribose ring of RNA is structurally constrained to increase the binding affinity for RNA or DNA. Thus, LNAs may be regarded as high affinity DNA or RNA derivatives. (Biochemistry vol 45, 7347-7355, 2006)
Antisense Mechanisms:
Antisense mechanism differs depending on types of AOs. RNAse H recognizes a duplex of mRNA with DNA, RNA, or PTO, and degrades the duplex portion of mRNA. Thus, the antisense activity of PTO is significantly amplified by RNAse H. In the meantime, RNAse H does not recognize a duplex of mRNA with PMO, PNA, or LNA. In other words, PMO, PNA and LNA must rely purely on the steric blocking of mRNA for their antisense activity. (Biochemistry vol 41, 4501-4510, 2002)
For oligonucleotides with the same binding affinity for mRNA, PTO should therefore show stronger antisense activity than PMO, PNA, and LNA. For steric block AOs such as PMO, PNA, and LNA, strong affinity for mRNA is desired for antisense activity.
Antisense Activity of PNA:
The binding affinity of PNA for mRNA would increase as the length of PNA increases to a certain point. However, the antisense activity of PNA doesn't seem to always increase to the length of PNA. There were cases that the antisense activity of PNA reached the maximum activity at 12 to 13-mer and decreases thereafter. (Nucleic acids Res. vol 32, 4893-4902, 2004) On the other hand, optimum antisense activity was reached with 15 to 18-mer PNAs against a certain mRNA, reflecting that the structural accessibility of the target binding site of the mRNA would be important. (Biochemistry vol 40, 53-64, 2001)
In many cases, PNAs have been reported to inhibit protein synthesis by ribosome at micromolar level under good cell penetrating conditions. (Science vol 258, 1481-85, 1992; Biochemistry vol 40, 7853-7859, 2001; Nucleic acids Res. vol 32, 4893-4902, 2004) However, PNAs targeting a highly accessible position of mRNA were found to show antisense activity at sub-micromolar level (Neuropeptides vol 38, 316-324, 2004; Biochemistry vol 40, 53-64, 2001) or even at sub-nanomolar level (Nucleic Acids Res. vol 36, 4424-4432, 2008) under good transfection conditions.
In addition to targeting a highly accessible site in mRNA, strong binding affinity of PNA for mRNA would be very required for good antisense activity. Unlike DNA, PTO, and LNA, the backbone of PNA is not charged. PNA tends to aggregate and become less suitable for binding to mRNA as its size increases. It is desired to improve PNA's binding affinity for mRNA without increasing the length of PNA. Incorporation of PNA monomers with a point charge would be beneficial in preventing PNA from aggregating.
Cell Penetration Strategies for PNA:
PNAs do not readily penetrate cell membrane and tend to show poor antisense activity unless properly transfected. In early years, the antisense activity of PNA was assessed by microinjection (Science vol 258, 1481-85, 1992) or electroporation (Biochemistry vol 40, 7853-7859, 2001). Microinjection and electroporation are invasive and inappropriate to be applied for therapeutic purposes. In order to improve the cell penetration, various strategies have been developed. (Adv. Drug Delivery Rev. vol 55, 267-280, 2003; Curr. Top. Med. Chem. vol 7, 727-737, 2007)
PNAs have been effectively delivered into cell by covalent incorporation of cell penetrating peptides (Neuropeptides vol 38, 316-324, 2004), lipofection following duplex formation with a complementary DNA (Biochemistry vol 40, 53-64, 2001), lipofection of PNAs with a covalently attached 9-aminoacridine (Nucleic Acids Res. vol 32, 2695-2706, 2004), lipofection of PNAs with covalently attached phosphonate anions (Nucleic Acids Res. vol 36, 4424-4432, 2008), and so on. Also cell penetration was improved by attaching to PNA a lipophilc moiety such as adamantane (Bioconjugate Chem. vol 10, 965-972, 1999) or amphiphilic group such as tetraphenyl phosphonium. (Nucleic Acids Res. vol 29, 1852-1863, 2001) Nevertheless, such a covalent modification is unlikely to increase the binding affinity for mRNA despite marked improvement in the cell penetration.
PNAs with a Covalently Attached CPP:
Cell penetrating peptides (CPPs) are polypeptides showing good cell penetration, and have multiple positive charges from arginine or lysine residues. To date many CPPs such as transportan, penetratin, NLS (nuclear localization signal), and Tat have been discovered. CPPs are known to efficiently carry a covalently attached cargo into cell. PNAs with a covalently attached CPP also showed good cell penetration.
Although some PNAs with a covalently attached CPP showed antisense IC50s around 100 nM (Neuropeptides vol 38, 316-324, 2004), micromolar antisense IC50s are rather prevalent for such PNAs.
PNAs with a covalently linked CPP are composed of two portions, the hydrophobic PNA domain and the positively charged CPP domain. Such a PNA tends to aggregate and be trapped in endosomes within cell, and would not be available for the antisense inhibition of protein synthesis. (Curr Top. Med. Chem. vol 7, 727-737, 2007; Nucleic Acids Res. vol 33, 6837-6849, 2005) Furthermore, such a covalently attached CPP hardly increases the binding affinity of PNA for mRNA.
PNAs with a Chiral Backbone:
There have been attempts to introduce a chiral substituent on the PNA backbone of 2-aminoethyl-glycine (Aeg). For example, the aqueous solubility of PNA was significantly improved by incorporating PNA monomer(s) with a backbone of 2-aminoethyl-lysine in place of Aeg. (Angew. Chem. Int. Ed. Engl. vol 35, 1939-1941, 1996)
By introducing the backbone of L-(2-amino-2-methyl)ethyl-glycine in place of Aeg, the binding affinity of PNA for DNA and RNA was significantly improved. A 10-mer PNA with all of the backbone of L-(2-amino-2-methyl)ethyl-glycine in place of 2-aminoethyl-glycine showed an increase of 19° C. and 10° C. in Tm against complementary DNA and RNA, respectively. Such an increase doesn't seem to be proportional to the number of substitution with L-(2-amino-2-methyl)ethyl-glycine, though. (J. Am. Chem. Soc. vol 128, 10258-10267, 2006)
GPNA:
The cell penetration of PNA was reported to be markedly improved by incorporating PNA monomers with a backbone of 2-aminoethyl-arginine in place of Aeg. (J. Am. Chem. Soc. vol 125, 6878-6879, 2003) Such PNAs have been termed ‘GPNA’ since they have guanidinium moiety on the backbone.
GPNAs with the backbone of 2-aminoethyl-D-arginine were reported to have stronger affinity for DNA and RNA than the corresponding GPNAs with that of 2-aminoethyl-L-arginine. (Chem. Commun. 244-246, 2005) For a 10-mer GPNA with 5 GPNA monomers with the backbone of 2-aminoethyl-D-arginine there was an increase of 7° C. in Tm (melting temperature) against complementary DNA compared to the corresponding unmodified PNA. (Bioorg. Med. Chem. Lett. vol 16, 4931-4935, 2006)
A 16-mer antisense GPNA against human EGFR-TK was reported to show antitumor activity upon ip (intra peritoneal) administration in athymic nude mice, although the in vitro antisense activity was not documented for the antisense GPNA in the prior art. (WO 2008/061091)
PNAs with Modified Nucleobase:
Like cases with DNA, nucleobase modifications have been pursued to improve PNA's affinity for nucleic acids.
PNAs with adenine replaced with 2,6-diaminopurine were evaluated for their affinity for complementary DNA or RNA. Substitution with 2,6-diaminopurine was found to elicit an increase of 2.5˜6° C. in Tm per replacement. (Nucleic Acids Res. vol 25, 4639-4643, 1997)

PNAs with cytosine replaced with 9-(2-aminoethoxy)phenoxazine were evaluated for their affinity for complementary DNA or RNA. A single substitution with 9-(2-aminoethoxy)phenoxazine elicited an increase of 10.7˜23.7° C. in Tm, although such an increase was markedly dependent on the nucleotide sequence. Nucleobase 9-(2-aminopropoxy)phenoxazine also induced a large increase in Tm. Due to a huge increase in Tm, PNA monomer with either 9-(2-aminoethoxy)-phenoxazine or 9-(2-aminopropoxy)phenoxazine as a cytosine replacement has been termed ‘G-clamp’. (Org. Lett. vol 4, 4395-4398, 2002) However, cell penetration data was not reported for PNAs with G-clamp(s).
PNAs with cytosine replaced with either 6-{2-(2-aminoethoxy)phenyl}-pyrrolocytosine or 6-{2,6-di(2-aminoethoxy)phenyl}pyrrolocytosine were evaluated for their affinity for complementary DNA or RNA. A single substitution with either 6-{2-(2-aminoethoxy)phenyl}pyrrolocytosine or 6-{2,6-di(2-aminoethoxy)-phenyl}pyrrolocytosine increased Tm by 3˜11.5° C. (J. Am. Chem. Soc. vol 130, 12574-12575, 2008) However, such PNAs were not evaluated for cell penetration.
Other Use of PNAs:
By tightly binding to a microRNA, PNA can inhibit the regulatory function of the microRNA, leading to an increase in the expression level of the protein(s) directly regulated by the microRNA. (RNA vol 14, 336-346, 2008) By tightly binding to a ribonucleoprotein such as telomerase, PNA can modulate the cellular function of the ribonucleoprotein. (Bioorg. Med. Chem. Lett. vol 9, 1273-78, 1999) By tightly binding to a certain portion of a gene in the nucleus, PNA can modulate the transcription level of the gene. (Biochemistry vol 46, 7581-89, 2007)
Since PNA tightly binds to DNA and RNA, and sensitively discriminates a single base pair mismatch, PNA would be suitable for high fidelity detection of single nucleotide polymorphism (SNP). Since PNA binds tightly to DNA and RNA with high sequence specificity, PNA may find various other therapeutic and diagnostic applications involving DNA or RNA. (FASEB vol 14, 1041-1060, 2000)